Structural polysaccharides comprise up to 90% of plant cell walls, and include cellulose and hemicellulose fractions as prominent resources renewable through photosynthesis. The hemicellulose fractions constitute from 22 to 30% of the dry weight of lignocellulosic biomass derived from wood and agricultural residues (Kuhad et al., 1997) and the quest for alternatives to petroleum has led to the search and discovery of microorganisms that can serve as biocatalysts for production of fuels and chemical feedstocks from renewable resources.
The major hemicellulose polymer in hardwoods and crop residues is methylglucuronoxylan (MeGAXn), a linear chain of β-1,4-linked D-xylopyranose residues regularly substituted with α-1,2-linked 4-O-methyl-D-glucuronopyranosyl residues. Variable substitutions on xylose residues may include 2′- and 3′-O-acetyl esters, as well as α-1,2- or α-1,3-linked L-arabinofuranosyl residues (Sunna et al., 1997). Additional substituents include O-feruloyl, and O-p-coumaroyl esters linked to hydroxyl groups on the arabinofuranosyl residues.
The natural processing of methylglucuronoxylans is catalyzed by the combined action of endoxylanases, α-glucuronidases, arabinosidases and esterases (Collins et al., 2005; Preston et al., 2003; Sunna et al., 1997). Xylanolytic bacteria secrete endoxylanases of glycohydrolase families GH5, GH10, and GH11 that catalyze the depolymerization of the xylan backbone with the generation of different products (Biely et al., 2000; Preston et al., 2003). The GH10 endoxylanases generate xylobiose, xylotriose, and the aldotetrauronate β-1,4-linked D-xylotriose substituted at the non reducing terminus with α-1,2-linked 4-O-methyl-D-glucuronate. Bacteria that secrete a GH10 endoxylanase may assimilate and metabolize all of the products derived from the depolymerization of MeGAXn. The utilization of the aldouronate requires the expression of genes encoding transporters, α-glucuronidase, and enzymes that convert xylooligosaccahrides to xylose. The glucuronate metabolism gene cluster in Geobacillus stearothermophilus T-6 includes genes that encode required activities, and has been well studied and defined with respect to structural and regulatory genes (Shulami et al., 1999; Shulami et al., 2007). Similar gene clusters have been found in several other bacteria as well (Nelson et al., 1999; Takami et al., 2000).
The isolation and characterization of an aggressively xylanolytic gram-positive endospore-forming bacterium, designated Paenibacillus sp. strain JDR-2, has been reported (St. John et al., 2006). This strain secretes a multimodular GH10 endoxylanase as a cell-anchored protein that catalyzes the depolymerization of MeGAXn (St. John et al., 2006). The rapid and complete utilization of MeGAXn without accumulation of the aldotetrauronate, methylglucuronoxylotriose (MeGAX3) in the medium implicated an efficient system for assimilation and complete metabolism of aldouronates. A structural gene, aguA, has been cloned from genomic DNA of Paenibacillus sp. strain JDR-2 and expressed in E. coli with the formation of a recombinant GH67 α-glucuronidase (AguA) that catalyzes conversion of MeGAX3 to methylglucuronate and xylotriose. This gene is followed by xynA2 encoding an intracellular GH10 endoxylanase catalytic domain (XynA2) that processes the xylotriose product generated by the action of AguA on MeGAX3. (Nong et al., 2005).
Both yeast and bacteria have been developed for the bioconversion of glucose derived from the cellulose fraction, and bacteria have been developed for the bioconversion of pentoses, principally xylose, from the hemicellulose fraction (Dien et al., 2003; Ingram et al., 1999; Kuhad et al., 1997). Pretreatment has relied on a combination of chemical and enzymatic hydrolytic procedures to solubilize the hemicellulose fraction and release fermentable xylose, and to depolymerize the cellulose to fermentable glucose. Pretreatment protocols are still being developed to provide cost-effective production of ethanol and other biobased products from these resources (Lloyd et al., 2005).